Determination of vitamin K encapsulated into lipid nanocarriers by dispersive liquid–liquid microextraction combined with liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry

Abstract Dispersive liquid–liquid microextraction was used in conjunction with liquid chromatography–atmospheric pressure chemical ionization–tandem mass spectrometry to quantitate vitamins K1 and K2 in vitamin‐fortified emulsions, and vital microextraction parameters were optimized using response surface methodology coupled with Box–Behnken design. Under optimal microextraction conditions, highly linear (R 2 > .999) calibration curves were obtained for both vitamins in a broad concentration range (1–1000 μg/L), and vitamin recoveries exceeded 90%. The detection and quantitation limits equaled 1.89 and 5.72 μg/L for vitamin K1, respectively, and 5.00 and 15.15 μg/L for vitamin K2, respectively. When applied to vitamin‐K‐loaded nanoemulsions and solid lipid nanoparticles, the developed method achieved excellent results, outperforming the currently employed Korean Food Code method, and therefore holding great promise for the quantitation of vitamin K in vitamin‐fortified food products.

possible and be compatible with a wide range of sample matrices (Vickers, 2017). Although traditional extraction methods, such as liquid-liquid extraction and solid-phase extraction, are widely used for sample pretreatment (Titato & Lanças, 2005), they exhibit certain drawbacks, which has inspired the development of alternative pretreatment methods like microextraction (Ibrahim et al., 2017;Kim et al., 2022).
In particular, dispersive liquid-liquid microextraction (DLLME, first reported in 2006; Rezaee et al., 2006) offers the advantages of high accuracy, low solvent consumption, short extraction time, and high enrichment factors (Viñas et al., 2014) and has been successfully used to extract highly and moderately lipophilic compounds like aromatic amines, polycyclic aromatic hydrocarbons, and pesticide residues (Andraščíková et al., 2013;Galuch et al., 2019;Liu et al., 2014;Özkan et al., 2019). In DLLME, the analyte is transferred from the aqueous phase (sample solution) to the organic phase (extraction solvent) with the help of a dispersive solvent, which reduces the polarity of the aqueous phase and increases the solubility of the target analyte in the organic phase (Farajzadeh et al., 2016;Lee et al., 2018Lee et al., , 2019. Most vitamin K analysis methods reported to date rely on highperformance liquid chromatography (HPLC) with different types of detection (e.g., ultraviolet, fluorescence, or electrochemical detection) after post-column derivatization (Dabre et al., 2011;Marinova et al., 2011). Among the detection techniques, fluorescence detection is used most commonly but is generally less sensitive than massspectrometric detection (Jäpelt & Jakobsen, 2016).
Herein, DLLME coupled with liquid chromatographyatmospheric-pressure chemical ionization-tandem mass spectrometry (LC-APCI-MS/MS) was tested as a method of vitamin K quantitation in vitamin-fortified foods, and vital DLLME parameters, such as NaCl content and extraction and dispersive solvent volumes, were optimized using response surface methodology coupled with Box-Behnken design. The optimized procedure was validated in terms of linearity, repeatability, accuracy, limit of detection (LOD), and limit of quantitation (LOQ) and used to quantitate vitamin K in correspondingly loaded lipid nanocarriers.

| Chemicals and reagents
Analytical standards of vitamins K1 and K2 were obtained from Sigma-Aldrich. Standard stock solutions (1 mg/L) were prepared in methanol and stored in amber glass vials at −20°C. Each standard solution was diluted with methanol to obtain seven aqueous working solutions (1, 5, 10, 50, 100, 500, and 1000 μg/L) that were used to construct standard calibration curves. HPLC-grade methanol, ethanol, acetonitrile, carbon tetrachloride, chloroform, and water were purchased from J.T. Baker. Carbon disulfide and acetone were purchased from Sigma-Aldrich.

| Sample preparation
Two types of vitamin-K-fortified samples (nanoemulsions [NEs] and solid lipid nanoparticles [SLNs]) were prepared. NEs were prepared by dissolving vitamin K (500 mg), lecithin (25 g), TW80 (25 g), and olive oil (50 g) in distilled water (900 ml). SLNs were prepared by dissolving vitamin K (250 mg), lecithin (2.5 g), TW80 (2.5 g), and palm oil (5 g) in water (90 ml) at 65°C for 5 min with subsequent rapid dispersion in an ice bath upon stirring at 90 g for 10 min using a Ultra Turrax (IKA Staufen GmbH, Germany) equipped with 18 G stirring probe. To reduce the matrix effect, the fortified samples were diluted 10,000fold with purified water to a final concentration of 50 μg/L prior to analysis.

| DLLME procedure
The extraction solvent (chloroform, 300 μl) was mixed with the dispersive solvent (methanol, 1 ml), and the mixture was rapidly injected into a conical glass tube containing a diluted sample (4 ml) and shaken for several seconds. The rapid injection of the solvent mixture produced a cloudy solution containing microdroplets of the extraction solvent dispersed in the aqueous phase (Lee et al., 2019).
The sample was subsequently centrifuged for 5 min at 1500 g, and the bottom phase (sedimented extraction phase microparticles) was collected and evaporated to dryness in a flow of nitrogen. The residue was reconstituted with acetonitrile (0.5 ml) and transferred to a vial using a syringe for analysis by LC-APCI-MS/MS.

| Experimental design and optimization by response surface methodology
The extraction and dispersive solvents were individually optimized before the application of the response surface model (RSM). Carbon tetrachloride, chloroform, dichloromethane, and carbon disulfide were tested as extraction solvents, while acetonitrile, methanol, and acetone were tested as dispersive solvents. After selecting the best dispersive and extraction solvents, we used Box-Behnken design (BBD) to construct a second-order RSM. The extraction solvent volume (X 1 ), dispersive solvent volume (X 2 ), and NaCl content (X 3 ) were selected as independent factors and studied at three levels ( Table 1).
The BBD included 15 experiments with three center points. The chromatographic peak area of vitamin K1 was used to evaluate extraction efficiency. The experimental data were fitted to a secondorder polynomial model, as shown in Equation (1).
where X i and X j are independent variables influencing response Y; β 0 , β i , β ii , and β ij are the regression coefficients for the intercept, linear, quadratic, and interaction terms, respectively; and k is the
The flow rate equaled 0.5 ml/min, and the total runtime was 6 min.
Vitamin K was determined in multiple reaction monitoring (MRM) mode at a capillary voltage of 1.0 kV, a cone voltage of 20 V, and extractor voltage of 3.0 V, a source temperature of 130°C, and a desolvation temperature of 450°C using nitrogen as a collision gas.
Masslynx™ version 4.1 software (Micromass, Waters) was used for instrumental control, data acquisition, and processing. The MRM parameters, retention times, and collision energies used for vitamin K analysis are listed in Table 2.

| Method validation
Diluted solutions of vitamin K standards were injected in triplicate, and calibration curves were constructed by plotting analyte peak area versus concentration. The LOD was measured as the concentration affording the weakest detectable peak with a signal to noise (S/N) ratio of 3, and the LOQ was determined as the lowest concentration allowing quantitative analysis and affording a peak with S/N = 10 (ICH, 2005). Precision was assessed by the intra-day and inter-day precisions and expressed as the relative standard deviation (RSD). Three replicate measurements were performed at three concentrations (5, 10 and 50 μg/L) using standard solutions.
Recoveries were evaluated as the ratio of the amounts of extracted analytes and initial analytes from a blank sample.

| Selection of extraction and dispersive solvents
The performances of extraction and dispersive solvents were assessed in terms of analyte recovery and extraction efficiency, respectively. As a result, chloroform and methanol were selected as optimal extraction and dispersive solvents, that is, as those affording the highest recovery and extraction efficiency (Figure 1).

| Method validation
The optimized method was validated in terms of accuracy, repeatability, LOD, and LOQ according to international guidelines (ICH, 2005). Linearity was evaluated using standard solutions of vitamin K with different concentrations (1, 5, 10, 50, 100, and 1000 μg/L), and the method was found to be highly linear (R 2 > .999). The LODs and LOQs were determined as 1.89 and 5.72 μg/L, respectively, for vitamin K1, and as 5.00 and 15.15 μg/L, respectively, for vitamin K2 (Table 4). Intra-and interday repeatabilities were evaluated for 5, 10, and 50 μg/L standard solutions and were found to be high, with RSD values lying below 5%. The recoveries obtained for samples fortified with 5, 10, and 50 μg/L vitamin K standard solutions exceeded 90% in all cases (

TA B L E 3 Analysis of variance data for
the quadratic response surface model

| Analysis of vitamin-fortified samples and performance comparison with Korean Food Code method
The developed method was used to quantitate vitamins K1 and K2 in two types of vitamin-fortified samples (NEs and SLNs) using the procedure described above. All experiments were performed in triplicate, with the results listed in

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C A L A PROVA L
This study does not involve any human or animal testing.